CYTOCHEMICAL CHARACTERIZATION OF OLIGOSACCHARIDE SIDE-CHAINS OF THE GLYCOPROTEINS OF RAT ZONA-PELLUCIDA - AN ULTRASTRUCTURAL-STUDY

Background: The zona pellucida (ZP), an extracellular matrix which sur rounds mammalian oocytes, is formed by different glycoproteins. Severa l studies have revealed that carbohydrate residues present in glycopro teins of ZP play a key role in the sperm-egg recognition. However, the origin and the biochemical composition of ZP remain to be completely resolved. Methods: ZP glycoproteins from rat ovarian follicles were in vestigated at light and electron microscopic level by the application of lectins conjugated to peroxidase, digoxigenin, and colloidal gold i n combination with enzyme and chemical treatment. A quantitative analy sis was also performed. Results: ZP shows reactivity to WGA, DSA, LFA, AAA, RCA I, and MAA. SBA and PNA showed a variable reactivity ranging from negative to strongly positive. A uniform pattern of binding thro ughout ZP was observed with DSA, Con A, AAA, MAA, and LFA. However, la beling by RCA I and SBA was higher in the outer ZP while PNA and WGA s howed a higher binding in the inner ZP. Lectin reactivity was detected in cortical granules, endoplasmic reticulum, Golgi apparatus, vesicle s, and multivesicular bodies of oocytes. Conclusions: ZP contained the terminal disaccharides Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, and Ga lNAc beta 1,3Gal and the trisaccharides Neu5Ac alpha 2, 3Gal beta 1,4G lcNAc, Neu5Ac-Gal beta 1,3GalNAc, and Neu5Ac-GalNAc beta 1,3Gal sequen ces. The occurrence of Fucose residues a 1,6 linked to the inner core region of N-linked glycoproteins of ZP was demonstrated by the use of several fucose-specific lectins. Methylation-saponification treatment in combination with lectin cytochemistry reveals that Gal, GalNAc, and polyl-lactosamine residues of rat ZP glycoproteins contain sulphated groups. The reactivity observed in ooplasmic vesicles was similar to t hat of ZP, thus suggesting that the oocyte is the site of synthesis of ZP glycoproteins. (C) 1994 Wiley-Liss, Inc.